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Metabolic reprogramming of metastatic breast cancer and melanoma by let-7a microRNA.

Serguienko A, Grad I, Wennerstrøm AB, Meza-Zepeda LA, Thiede B, Stratford EW, Myklebost O, Munthe E - Oncotarget (2015)

Bottom Line: Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes.To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin.Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

ABSTRACT
Let-7 microRNAs (miRNAs) are highly conserved well-established promoters of terminal differentiation that are expressed in healthy adult tissues and frequently repressed in cancer cells. The tumor suppressive role of let-7 in a variety of cancers in vitro and in vivo has been widely documented and prompted these miRNAs to be candidate genes for miRNA replacement therapy. In this study we described a new role of let-7a in reprogramming cancer metabolism, recently identified as a new hallmark of cancer. We show that let-7a down-regulates key anabolic enzymes and increases both oxidative phosphorylation and glycolysis in triple-negative breast cancer and metastatic melanoma cell lines. Strikingly, the accelerated glycolysis coexists with drastically reduced cancer features. Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes. To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin. In both cancer types let-7a increased cell sensitivity to doxorubicin. Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway. We thus have demonstrated that let-7a plays a prominent role in regulating energy metabolism in cancer cells, further expanding its therapeutic potential.

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Related in: MedlinePlus

Let-7a affects key anabolic enzymes and cell-cycle progression in MDA-MB-231A, Protein levels of G6PDH, TYMS, FASN, AASDHPPT, and SCD on day 3 post transfection. WBs quantification is shown in the Table 1. B, cell-cycle was analyzed by flow cytometry using Hoechst 33342 staining on day 3 post transfection, n=6, SD.
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Figure 2: Let-7a affects key anabolic enzymes and cell-cycle progression in MDA-MB-231A, Protein levels of G6PDH, TYMS, FASN, AASDHPPT, and SCD on day 3 post transfection. WBs quantification is shown in the Table 1. B, cell-cycle was analyzed by flow cytometry using Hoechst 33342 staining on day 3 post transfection, n=6, SD.

Mentions: The effect of let-7a on global transcript levels was determined using microarray. Rank product analysis was employed to identify differentially expressed genes. The analysis revealed 873 probes, representing 754 genes, with significant changes in gene expression between let-7a transfected and negative control cells. Of these, 357 genes were down-regulated whereas 397 genes were up-regulated. Functional enrichment analysis of microarray data (GEne SeT AnaLysis-GESTALT-Toolkit) identified metabolic pathways to be highly enriched (Table S1). To investigate differences at the protein level we used SILAC-base proteome-wide analysis. We identified 37 proteins with significantly changed levels upon let-7a transfection. Of these, 27 proteins were down-regulated, and 10 proteins were up-regulated (Table S2). Comparison of the mRNA and protein changes revealed that 54% of the differentially expressed proteins were also changed in the same direction at the mRNA level (Fig. S2A). Enrichment analysis of changed proteins also showed metabolic pathways as the first group in the top ten list (Table S1). Among metabolic genes down-regulated by let-7a at the transcriptional and/or protein level we identified key enzymes of anabolic pathways, namely G6PD, inosine monophosphate dehydrogenase (IMPDH2), FASN, stearoyl-CoA desaturase (SCD) and 4-phosphopantetheinyl transferase (AASDHPPT) (Table 1). Protein level changes were further validated by Western blot analysis (Fig. 2 and Table 1). Differentially expressed mRNAs and proteins were compared with the list of predicted let-7a targets and revealed that among anabolic genes only SCD is a predicted let-7 target (Fig. S2B). Since these enzymes are essential for macromolecular synthesis underlying cell cycle progression, we performed a cell-cycle analysis. The detection of Hoechst fluorescence by flow cytometry revealed that the fraction of cells in S-phase is reduced by 50% in let-7a transfected cells compared to the negative control (Fig. 2B).


Metabolic reprogramming of metastatic breast cancer and melanoma by let-7a microRNA.

Serguienko A, Grad I, Wennerstrøm AB, Meza-Zepeda LA, Thiede B, Stratford EW, Myklebost O, Munthe E - Oncotarget (2015)

Let-7a affects key anabolic enzymes and cell-cycle progression in MDA-MB-231A, Protein levels of G6PDH, TYMS, FASN, AASDHPPT, and SCD on day 3 post transfection. WBs quantification is shown in the Table 1. B, cell-cycle was analyzed by flow cytometry using Hoechst 33342 staining on day 3 post transfection, n=6, SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385863&req=5

Figure 2: Let-7a affects key anabolic enzymes and cell-cycle progression in MDA-MB-231A, Protein levels of G6PDH, TYMS, FASN, AASDHPPT, and SCD on day 3 post transfection. WBs quantification is shown in the Table 1. B, cell-cycle was analyzed by flow cytometry using Hoechst 33342 staining on day 3 post transfection, n=6, SD.
Mentions: The effect of let-7a on global transcript levels was determined using microarray. Rank product analysis was employed to identify differentially expressed genes. The analysis revealed 873 probes, representing 754 genes, with significant changes in gene expression between let-7a transfected and negative control cells. Of these, 357 genes were down-regulated whereas 397 genes were up-regulated. Functional enrichment analysis of microarray data (GEne SeT AnaLysis-GESTALT-Toolkit) identified metabolic pathways to be highly enriched (Table S1). To investigate differences at the protein level we used SILAC-base proteome-wide analysis. We identified 37 proteins with significantly changed levels upon let-7a transfection. Of these, 27 proteins were down-regulated, and 10 proteins were up-regulated (Table S2). Comparison of the mRNA and protein changes revealed that 54% of the differentially expressed proteins were also changed in the same direction at the mRNA level (Fig. S2A). Enrichment analysis of changed proteins also showed metabolic pathways as the first group in the top ten list (Table S1). Among metabolic genes down-regulated by let-7a at the transcriptional and/or protein level we identified key enzymes of anabolic pathways, namely G6PD, inosine monophosphate dehydrogenase (IMPDH2), FASN, stearoyl-CoA desaturase (SCD) and 4-phosphopantetheinyl transferase (AASDHPPT) (Table 1). Protein level changes were further validated by Western blot analysis (Fig. 2 and Table 1). Differentially expressed mRNAs and proteins were compared with the list of predicted let-7a targets and revealed that among anabolic genes only SCD is a predicted let-7 target (Fig. S2B). Since these enzymes are essential for macromolecular synthesis underlying cell cycle progression, we performed a cell-cycle analysis. The detection of Hoechst fluorescence by flow cytometry revealed that the fraction of cells in S-phase is reduced by 50% in let-7a transfected cells compared to the negative control (Fig. 2B).

Bottom Line: Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes.To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin.Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

ABSTRACT
Let-7 microRNAs (miRNAs) are highly conserved well-established promoters of terminal differentiation that are expressed in healthy adult tissues and frequently repressed in cancer cells. The tumor suppressive role of let-7 in a variety of cancers in vitro and in vivo has been widely documented and prompted these miRNAs to be candidate genes for miRNA replacement therapy. In this study we described a new role of let-7a in reprogramming cancer metabolism, recently identified as a new hallmark of cancer. We show that let-7a down-regulates key anabolic enzymes and increases both oxidative phosphorylation and glycolysis in triple-negative breast cancer and metastatic melanoma cell lines. Strikingly, the accelerated glycolysis coexists with drastically reduced cancer features. Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes. To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin. In both cancer types let-7a increased cell sensitivity to doxorubicin. Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway. We thus have demonstrated that let-7a plays a prominent role in regulating energy metabolism in cancer cells, further expanding its therapeutic potential.

Show MeSH
Related in: MedlinePlus